Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

<p>Abstract</p> <p>Background</p> <p>In a traditional electrophoresis mobility shift assay (EMSA) a ³²P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence.</p> <p>Results</p> <p>We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (<it>Alu</it>I, <it>Bsu</it>RI, <it>Tru</it>I, etc), separation of low and high molecular weight fractions of resultant DNA fragments, ³²P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (<it>p75NTR</it>) gene.</p> <p>Conclusion</p> <p>The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis.</p

Suguhormoonide östradiooli ja progesterooni koespetsiifiline roll inimese endomeetriumis ja rinnanäärmes

Artiklis on antud ülevaade östradiooli (E2) ja progesteroon (P4) olulisusest inimese endomeetriumis ja rinnanäärmes ning kirjeldatud nende hormoonide varjupoolt seoses viljatuse ja rinnavähiga. On oluline mõista, et samadel hormoonidel võib erinevates kudedes olla erinev või lausa vastupidine efekt. Vaatamata laialdastele uuringutele on paljud küsimused seoses suguhormoonide regulatsiooniga seni veel vastamata. Rinnavähk ja sellega seonduv on tänapäeval üks enim uuritud valdkondi, kuid see on naiste seas siiani enim elusid nõudnud vähitüüp. Üha rohkem paare vajab kunstliku viljastamise protseduuride (IVF) kliinikute abi lapse saamisel ning jätkuvalt otsitakse võimalusi, kuidas ravi tulemuslikkust parandada ja võimalikke kõrvalmõjusid vähendada. Autorid tahaksid rõhutada kogu genoomi sekveneerimismeetodite olulisust, sest need meetodid võimaldavad uurida korraga kogu inimese genoomi. Vastav info võimaldab tulevikus muu hulgas ennustada, kuidas üks või teine ravim või preparaat konkreetsele inimesele mõjub. Eesti Arst 2012; 91(4):182-18

The Influence of Different Maternal Microbial Communities on the Development of Infant Gut and Oral Microbiota

Very few studies have analyzed how the composition of mother's microbiota affects the development of infant's gut and oral microbiota during the first months of life. Here, microbiota present in the mothers' gut, vagina, breast milk, oral cavity, and mammary areola were compared with the gut and oral microbiota of their infants over the first six months following birth. Samples were collected from the aforementioned body sites from seven mothers and nine infants at three different time points over a 6-month period. Each sample was analyzed with 16S rRNA gene sequencing. The gut microbiota of the infants harbored distinct microbial communities that had low similarity with the various maternal microbiota communities. In contrast, the oral microbiota of the infants exhibited high similarity with the microbiota of the mothers' breast milk, mammary areola and mouth. These results demonstrate that constant contact between microbial communities increases their similarity. A majority of the operational taxonomic units in infant gut and oral microbiota were also shared with the mothers' gut and oral communities, respectively. The disparity between the similarity and the proportion of the OTUs shared between infants' and mothers' gut microbiota might be related to lower diversity and therefore competition in infants' gut microbiota.Peer reviewe

Genes targeted by the estrogen and progesterone receptors in the human endometrial cell lines HEC1A and RL95-2

<p>Abstract</p> <p>Background</p> <p>When the steroid hormones estrogen and progesterone bind to nuclear receptors, they have transcriptional impact on target genes in the human endometrium. These transcriptional changes have a critical function in preparing the endometrium for embryo implantation.</p> <p>Methods</p> <p>382 genes were selected, differentially expressed in the receptive endometrium, to study their responsiveness of estrogen and progesterone. The endometrial cell lines HEC1A and RL95-2 were used as experimental models for the non-receptive and receptive endometrium, respectively. Putative targets for activated steroid hormone receptors were investigated by chromatin immunoprecipitation (ChIP) using receptor-specific antibodies. Promoter occupancy of the selected genes by steroid receptors was detected in ChIP-purified DNA by quantitative PCR (qPCR). Expression analysis by reverse transcriptase (RT)-PCR was used to further investigate hormone dependent mRNA expression regulation of a subset of genes.</p> <p>Results</p> <p>ChIP-qPCR analysis demonstrated that each steroid hormone receptor had distinct group of target genes in the endometrial cell lines. After estradiol treatment, expression of estrogen receptor target genes predominated in HEC1A cells (n = 137) compared to RL95-2 cells (n = 35). In contrast, expression of progesterone receptor target genes was higher in RL95-2 cells (n = 83) than in HEC1A cells (n = 7) after progesterone treatment. RT-PCR analysis of 20 genes demonstrated transcriptional changes after estradiol or progesterone treatment of the cell lines.</p> <p>Conclusions</p> <p>Combined results from ChIP-qPCR and RT-PCR analysis showed different patterns of steroid hormone receptor occupancy at target genes, corresponding to activation or suppression of gene expression after hormone treatment of HEC1A and RL95-2 cell lines.</p

Στρατηγικές αποτιμήσεις του εκπαιδευτικού έργου στη χώρα μας στην Πρωτ/θμια και Δευτ/θμια εκπαίδευση, στο διάστημα 1995-2004 : (εργαλεία και μέσα)

BACKGROUND: In the last years, many olive plantations in southern Spain have been mediated by the use of self-rooted planting stocks, which have incorporated commercial AMF during the nursery period to facilitate their establishment. However, this was practised without enough knowledge on the effect of cropping practices and environment on the biodiversity of AMF in olive orchards in Spain. METHODOLOGY/PRINCIPAL FINDINGS: Two culture-independent molecular methods were used to study the AMF communities associated with olive in a wide-region analysis in southern Spain including 96 olive locations. The use of T-RFLP and pyrosequencing analysis of rDNA sequences provided the first evidence of an effect of agronomic and climatic characteristics, and soil physicochemical properties on AMF community composition associated with olive. Thus, the factors most strongly associated to AMF distribution varied according to the technique but included among the studied agronomic characteristics the cultivar genotype and age of plantation and the irrigation regimen but not the orchard management system or presence of a cover crop to prevent soil erosion. Soil physicochemical properties and climatic characteristics most strongly associated to the AMF community composition included pH, textural components and nutrient contents of soil, and average evapotranspiration, rainfall and minimum temperature of the sampled locations. Pyrosequencing analysis revealed 33 AMF OTUs belonging to five families, with Archaeospora spp., Diversispora spp. and Paraglomus spp., being first records in olive. Interestingly, two of the most frequent OTUs included a diverse group of Claroideoglomeraceae and Glomeraceae sequences, not assigned to any known AMF species commonly used as inoculants in olive during nursery propagation. CONCLUSIONS/SIGNIFICANCE: Our data suggests that AMF can exert higher host specificity in olive than previously thought, which may have important implications for redirecting the olive nursery process in the future as well as to take into consideration the specific soils and environments where the mycorrhized olive trees will be established

Co-expression of TrkB and TrkC receptors in CNS neurones suggests regulation by multiple neurotrophins

Using double-label in situ hybridization, we have explored the possibility that individual CNS neurones can co-express mRNAs for the high-affinity receptors of brain-derived neurotrophic factor and neurotrophin-3, TrkB and TrkC, respectively. The vast majority of TrkC mRNA-containing neurones in the hippocampal formation and cerebral cortex were also labelled for TrkB mRNA. Cells expressing only TrkB or TrkC mRNA were very scarce in these regions, representing < 15% of all labelled cells. These findings suggest that the same cortical or hippocampal neurone can be regulated by several members of the neurotrophin family, which may be important both during development and in response to physiological activity and pathological conditions

Influence of Edaphic, Climatic, and Agronomic Factors on the Composition and Abundance of Nitrifying Microorganisms in the Rhizosphere of Commercial Olive Crops

17 páginas, 4 tablas, 4 figuras.The microbial ecology of the nitrogen cycle in agricultural soils is an issue of major interest. We hypothesized a major effect by farm management systems (mineral versus organic fertilizers) and a minor influence of soil texture and plant variety on the composition and abundance of microbial nitrifiers. We explored changes in composition (16S rRNA gene) of ammonia-oxidizing archaea (AOA), bacteria (AOB), and nitrite-oxidizing bacteria (NOB), and in abundance of AOA and AOB (qPCR of amoA genes) in the rhizosphere of 96 olive orchards differing in climatic conditions, agricultural practices, soil properties, and olive variety. Majority of archaea were 1.1b thaumarchaeota (soil crenarchaeotic group, SCG) closely related to the AOA genus Nitrososphaera. Most AOB (97%) were identical to Nitrosospira tenuis and most NOB (76%) were closely related to Nitrospira sp. Common factors shaping nitrifiers assemblage composition were pH, soil texture, and olive variety. AOB abundance was positively correlated with altitude, pH, and clay content, whereas AOA abundances showed significant relationships with organic nitrogen content and exchangeable K. The abundances of AOA differed significantly among soil textures and olive varieties, and those of AOB among soil management systems and olive varieties. Overall, we observed minor effects by orchard management system, soil cover crop practices, plantation age, or soil organic matter content, and major influence of soil texture, pH, and olive tree variety.This research was supported by grants P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’-Junta de Andalucía, ‘Interprofesional del Aceite de Oliva Español-Fundación CITOLIVA’ and AGL-2012-37521 from the Spanish Office of Science (MINECO) to BBL,DARKNESS CGL2012-32747 (MINECO) to EOC, and Fondo Europeo de Desarrollo Regional (FEDER).Peer reviewe